Biodiversity Of Free-Living Dizotrophs Isolated From Soils In Liusha River Valley

Fifty-three free-living Dizotrophs strains isolated from soils at different altitude inLiusha river valley were analyzed by phenotypic features, BOX-PCR, RAPD and 16SrRNA PCR-RFLP to reveal their phenotypic and genotypic diversity.The results of phenotypic features showed that the tested strains had differentphysiological and biochemical characteristics, including carbon and nitrogen sourcesusage, antibiotic resistance, chemical drug tolerance, salt and corrosion resistance, etc.and there were 8 strains could grow on the medium containing 4% NaCl, 4 strains couldgrow in the environment of pH4.0, and 13 strains could grow under 4℃conditions.Furthermore, strain 1262 was the only strain using ammonium molybdate as solenitrogen source, and could tolerate 6 kinds of antibiotics tested at concentration of300ug/mL.Dendrogram of numerical taxonomy showed that all the tested strains were dividedinto 17 independent groups at the boundary of 82.6% similarity, and 7 individual groupswere included; group 5 and group 12 were the two largest among these groups, and wereformed by 9 and 14 strains, respectively; these strains distributed in the habitats at thealtitude from 722m～1440m and 747m～1800m, and in purple soil, yellow soil, andpaddy soil, and so on, which suggested that strains in these two groups were thedominant ones in these areas. There was no obvious correlation between the phenotypicgroups and soil types and altitude strains distributed, which demonstrated the phenotypicdiversity among the strains was existed.Abundant bands were amplified in analysis of BOX-PCR, which could be used toreveal the existence of genetic diversity. All the strains clustered into 10 different geneticgroups at the similarity of 68%, except strain 1051 grouped individually, the other strainsaccumulated 9 genetic groups with 2～14 strains for each.Six RAPD primers were chosen for RAPD analyzing, each primer was produced 2～17 bands separately. The results of RAPD analysis indicated that all the strains weredivided into 9 groups at the similarity of 70%, and 32 strains grouped into group 2, 4 and5; there were only 2 strains in group 8, and the others were contained 3-6 strains, respectively. The results also showed that RAPD revealed the genetic diversity amongstrains effectively.The 16S rDNA PCR-RFLP analysis resulted in 27 different genotypes by 4 kinds ofendonuclease (MspⅠ, HaeⅢ, HinfⅠand AluⅠ), there were 16S rDNA genotypes formed,and genotype 6 and 12 had 10 and 5 strains respectively, and the other genotypes contained2-3 strains, which indicated that these strains had great diversity in phylogeny. The 16SrDNA PCR-RFLP dendrogram showed that there were 8 genetic groups formed at 78.6%similarity level, and group 4 and 6 were the largest two groups, and were constructed by 11and 14 strains, respectively.Overall, the analysis of phenotypic and genotypic diversity was good agreementwith each other, which approved the research results were credible and stable. Forexample, the group 2, 4 and 10 of BOX-PCR were good agreement with group 5, 13 and12 of numerical analysis, and the group 3, 4 and 8 of RAPD were equal to group 6, 12and 8 of numerical analysis, and the group 5, 6 and 8 were identical with group 6, 12 and8 of numerical analysis, which indicated the 4 methods used could reveal the biodiversityamong the tested free-living nitrogen fixation strains.Since no reference strain was used in the study, the 16S rDNA full sequence andDNA-DNA hybridization should be performed to determine the exact taxonomic positionand phylogeny of free-living Dizotrophs.